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Oct 2, 1991 - Epidermolysis bullosa, a rare genodermatosis. is characterized by increased skin fragility manifest as blistering and sometimes accompanied ...
Histopiflioloyy 1992. 20, 237-241

Expression of mutant p53 gene in squamous carcinoma arising in patients with recessive dystrophic epidermolysis bullosa S.D.SLATER. J.A.McGRATH*, C.HOBBS, R.A.J. EADY* & P.H.McKEE Department of Histopathology. St Thornas' Hospital Medical School and *Institute of Derrnatology arid St John's Hospital Derniatology Centre, St Thomas' Hospital. London, U K Date of submission h August 199 1 Accepted for publication 2 October 1991

SLATER S . D . , MCGRATH J . A . , HOBBS C . , E A D Y R . A . J . & MCKEE P . H .

(1992) Histopathology 20, 237-241

Expression of mutant p53 gene in squamous carcinoma arising in patients with recessive dystrophic epidermolysis bullosa Epidermolysis bullosa, a rare genodermatosis. is characterized by increased skin fragility manifest as blistering and sometimes accompanied by scarring. The latter is particularly severe in the recessive dystrophic variant and may be complicated by the development of squamous carcinoma in up to 3 0 % of patients. We have studied 2 3 such tumours in six patients with this variant, with an anti-serum to p5 3 protein. Twenty-six per cent of the squamous Carcinomas labelled positively for tnutant-type p5 3 protein. This low figure, however, reflects the large number of welldifferentiated tumours in this series. where 1 4 out of 15 were negative. In the moderate to poorly differentiated examples the positivity rate was 63%. Of the three patients in the latter category, one has died from disseminated tuniour and another has widespread metastases. The findings support the hypothesis that mutant p5 3 protein expression correlates with poorer tumour differentiation. They also suggest a possible correlation between p5 3 protein expression and tumour behaviour. Keywords: mutant p 5 3 protein, squamous carcinoma. epidermolysis bullosa

Introduction Epidermolysis bullosa consists of a group of rare, inherited muco-cutaneous diseases characterized by increased skin fragility of varying degree, manifest as blistering and erosions often accompanied by The recessive dystrophic variant is characterized by epidermo-dermal separation through the sub-lamina densa zone%.It is due, at least in part, to a deficiency of type VII ~ o l l a g e nassociated ~,~ with reduction or absence of the anchoring fibrils6-' vital for dermo-epidermal junction integrity. Recessive dystrophic epidermolysis bullosa is associated with very marked dermal fibrosis, and contractures are a common clinical finding'. Patients are at considerable risk of developing multiple synchronous and metachronous cutaneous squamous cell carcinomas which are associated with considerable Address for correspondence: Dr S.D.Slater. Department of Histopathology, Royal Postgraduate Medical School. Hammersmith Hospital. London W 1 2 OHS. UK.

morbidity and mortality"'. In early proliferative lesions the histological features are often equivocal, making a diagnosis of malignancy impossible despite a high index of suspicion. These borderline lesions are obviously important as many may have malignant potential and the capacity for metastasis. The Department of Cell Pathology based at St John's Institute of Dermatology, St Thomas' Hospital has operated a referral centre for 12 years for patients with epidermolysis bullosa congenita. Over 300 patients with all types of epidermolysis bullosa have been studied during this time. At least 35 of these have the severe recessive dystrophic variant and 11 ( 3 1%)have developed cutaneous malignancies"'. The diagnosis and effective early management of these patients might be greatly improved if a marker of neoplastic biological potential could be identified. The pathogenesis of cancer is a multi-step process which progresses as key regulating genes, usually tumour-suppressor and/or proto-oncogenes, sustain 237

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damage”. p53, a nuclear phosphoprotein, is believed to act as a tumour-suppressor gene controlling entry into the S phase of the cell cycleI2. Mutation of the normal (wild-type) p53 gene produces an abnormal mutant p5 3 protein which appears to confer a growth advantage on the cell line13. This is associated with a dramatic prolongation of the half-life of the p53 protein and an altered c~nformation’~. Thus, in contrast to the wildtype p53 present in normal tissues and undetectable by current immunocytochemical methodology, the mutant form may be easily and specifically indentified using routine immunoperoxidase techniques. Accumulated mutant p5 3 protein has been identified immunohistochemically in a wide range of human malignancies, including breast, lung, colon, ovary, stomach, pancreas, bladder and testicular carcinoma, soft tissue sarcomas and malignant melanoma’ 5 - 1 7. l‘he mutant protein is confined to neoplasms with malignant potential and is not thought to be present in normal tissues’ i . It is normally identified within nuclei although, rarely. cytoplasmic labelling has been recorded”. It appears to be one of the commonest mutations in carcinomas, with positivity rates of 5087%’I j 7 . As the mutation appears to be a late genetic change in cancer evolution, its presence may be a useful readily identifiable marker of malignancy with direct clinical application’ ,’(’. The purpose of this study was to test the hypothesis that immunohistochemically determined p5 3 mutation in squamous cell carcinomas from patients with epidermolysis bullosa correlates with the degree of differentiation and therefore potential behaviour of the tumour.

Materials and methods All of the squamous carcinomas arising in patients with recessive dystrophic epidermolysis bullosa were drawn from the tiles of the departments of histopathology of St Thomas’ Hospital and St John’s Hospital for Diseases of the Skin over the period from 1983 to the present day. Eleven patients were identified. From these, 2 3 tumours arising in six patients had residual formalin-fixed paraffin-embedded material available for study. The tumours were reviewed histologically and graded by one observer-P.H.M. The iinmunohistochemistry was performed using the rabbit polyclonal anti-p53 antiserum CM-1 (a gift from Professor D.Lane, The Cancer Research Campaign Laboratories, Department of Biochemistry. University of Dundee, UK), raised against recombinant human p5 324. This polyclonal antibody was chosen because it can be used on paraffin-embedded tissues. The alternative

monoclonal antisera 1801/240 can only be used on unfixed material. After dewaxing, the tissue sections were immersed in 0.5% HzOl in methanol for 1 5 min followed by normal swine serum 1/20 (Sera Lab) for 10 min, to block endogenous peroxidase activity and non-specific binding of antibody respectively. The primary antibody was used at dilutions of 1 : 1000-1 : 32 000 and the sections were incubated overnight at 4°C. A streptavidin-biotin peroxidase system (Dakopatts, UK) was used for antibodyantigen complex detection and diaminobenzidine was used as the chromogen. To intensify the reaction product a standard hexamine silver solution was then appliedIX. The sections were then lightly counterstained with Mayer’s haematoxylin for 10 seconds. A known p53 mutant-type protein positive breast carcinoma was run concurrently in each experiment. Negative controls included exclusion of the primary antiserum and exclusion of the streptavidin-biotin peroxidase system.

Results In occasional tumours the use of low dilutions of p53 (1: 1000 and 1: 2000) was associated with labelling of vascular smooth muscle cytoplasm and eccrine sweat gland myoepithelial cells. Further dilution to 1: 3 2 000, however, resulted in abolition of this staining but had no effect on tumour cell nuclear labelling. The majority of tumours (16: 65%) were welldifferentiated squamous carcinomas showing abundant keratinization. Most of these (15/16) expressed no mutant p53 protein. However, in one tumour isolated nuclei present in the advancing border stained positively (Figure 1).One tumour, although largely well-differentiated showed areas of moderate differentiation. In the latter areas immunolabelling for p53 protein of the tumour nuclei was very marked and was present in most of the cells (Figure 2). The transformation from immunonegative well-differentiated to immunopositive moderately differentiated tumour was striking. Three tumours showed variable differentiation, ranging from well to moderate to poor. Two of these labelled positively for p5 3 protein. Two tumours were uniformly poorly differentiated. One of these showed marked positivity, affecting about 50% of nuclei; the other was immunonegative. A further tumour was a poorly differentiated spindle cell variant in which isolated nuclei were positively labelled. In two patients with multiple tumours there were differences between tumours. One patient had 11 welldifferentiated tumours which were all p5 3 negative and one poorly differentiated tumour which was positive. The second patient had two well-differentiated tumours

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Figure 1. Well-differentiated squanious carcinoma: a nuclear labelling for p 5 3 protein at the advancing border, b negative control.

which were negative and one moderately differentiated tumour which was positive.

Discussion Twenty-six percent of the recessive dystrophic epiderniolysis bullosa related tumours labelled for mutant p53. In general, positivity was limited to the moderately or poorly differentiated tumours; however. positively labelled nuclei were identified along the advancing edge of one otherwise well-differentiated tumour. The p5 3 mutation appears to be one ofthe commonest mutations in carcinomas with positivity rates of 5087%,for epithelial tumours’ 5-1 The frequency of occurrence of immunocytochemically detectable p5 3 appears to be determined by the type of tumour studied, the specific mutation present and the sensitivity and specificity of the technique. It has been postulated that the role of p5 3 may vary in different tumour categories. p53 mutations have not

’.

been found in embryonal tumours such as retinoblastoma whereas in the Li-Fraumeni syndrome, a n inherited cancer syndrome, all of the cells of affected members have one defective p53 gene”. The epithelial tumours of later life probably involve more changes than a single mutation of the p5 3 allele. The type of damage sustained by the p53 gene may also influence immunocytochemical detection of the mutant protein, whilst still rendering it non-functional. Some mutations do not produce over-accumulation of the mutant protein’ I . Presently available detection methods are not sufficiently sensitive to detect low levels of mutant protein“. Some tumours have p5J gene mutations which completely abolish expression of the full length protein. These mutations are also not discernible immunocytochemically”. Specific mutations in p5 3 may be produced by certain carcinogens”. Thus, it is possible that the mutagenic agents may influence the type and amount of mutant p53 protein present. In our study the frequency of p5 3 positivity was low.

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role of mutant p53 in tumour progression may be more than an absence of function as a tumour suppressor and may be the result of some intrinsic positive growth promoting function, acting in some circumstances as a dominant transforming oncogene". On purely morphological grounds poor histological grade and mutant p5 3 detection were associated findings in our study. Of the three patients with moderate to poorly differentiated or spindle cell neoplasms, one has died and one has widespread metastatic disease. In the third patient insufficient time has elapsed since diagnosis to allow a meaningful follow-up. The three patients with welldifierentiated tumours are all alive and free from metastases. Although the numbers are small, these findings suggest a possible correlation between mutant-type p5 3 expression and tumour behaviour, and support the need for further study.

Acknowledgements We are indebted to Professor D.J,ane, The Cancer Research Campaign Laboratories, Department of Biochemistry, llniversity of Dundee. who generously supplied the p53 antiserum. We are grateful to Professor P.Hall. Department of Histopathology, St Thomas' Hospital Medical School for his helpful advice and to Miss J.Hauxwell for her hard work and patience in the preparation of this manuscript. J.A.M. is currently receiving a research fellowship from the Dystrophic Epidermolysis Bullosa Research Association (DEBRA). Figure 2. Moderately differentiated squamous carcinoma showing widespread nuclear labelling for p5 3 protein.

References However. the sample group of tumours consisted of 1 6/ 2 3 well-differentiated tumours and 7/2 3 moderately to poorly differentiated tumours. p5 3 positivity was confined to histologically more aggressive lesions, with 63'x of these staining positively. In a previous study of colorectal neoplasms, the morphological transition between adenonia and carcinoma within a particular tumour correlated with the appearance of p5 3 mutant protein"'. It has been suggested by other authors that. as wild-type p5 3 has a rate limiting role in proliferation and as the mutation appears late in tumour development. it might be associated with rapid proliferation, poor differentiation or associated bad prognostic signsi4.Certainly in breast carcinoma such an association has been reported with oestrogen receptor negativity, epidermal growth factor positivity and neu expression-all poor prognostic features2'-13.It has been suggested that the

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